The invention relates to cell death diseases.
Neurodegenerative diseases include familial and sporadic amyotrophic lateral sclerosis (FALS and ALS, respectively), familial and sporadic Parkinson""s disease, Huntington""s disease, familial and sporadic Alzheimer""s disease, olivopontocerebellar atrophy, multiple system atrophy, progressive supranuclear palsy, diffuse lewy body disease, corticodentatonigral degeneration, progressive familial myoclonic epilepsy, strionigral degeneration, torsion dystonia, familial tremor, Gilles de la Tourette syndrome, and Hallervorden-Spatz disease. Most of the diseases are typified by onset during the middle adult years and lead to rapid degeneration of specific subsets of neurons within the neural system, ultimately resulting in premature death. There is no known cure nor is there an effective therapy to slow the progression for any of the stated diseases.
Amyotrophic lateral sclerosis (ALS) is the most commonly diagnosed progressive motor neuron disease. The disease is characterized by degeneration of motor neurons in the cortex, brainstem and spinal cord (Principles of Internal Medicine, 1991 McGraw-Hill, Inc., New York; Tandan et al. Ann. Neurol, 18:271-280, 419-431, 1985). Generally, the onset is between the third and sixth decade, typically in the sixth decade. ALS is uniformly fatal, typically within five years (Kurland et al., Proc Staff Meet Mayo Clin, 32:449-462, 1957). The cause of the disease is unknown and ALS may only be diagnosed when the patient begins to experience asymmetric limb weakness and fatigue, localized fasciculation in the upper limbs and/or spasticity in the legs which typifies onset.
In ALS the neurons of the cerebral cortex and anterior horns of the spinal cord, together with their homologues in some of the motor nuclei of the brain stem, are affected. The class of neurons affected is highly specific: motor neurons for ocular motility and sphincteric motor neurons of the spinal cord remain unaffected until very late in the disease. Although death occasionally results shortly after the onset of the symptomatic disease, the disease generally ends with respiratory failure secondary to profound generalized and diaphragmatic weakness.
About 10% of ALS cases are inherited as an autosomal dominant trait with high penetrance after the sixth decade (Mulder et al. Neurology, 36:511-517, 1986; Horton et al. Neuroloay, 26:460-464, 1976). In almost all instances, sporadic and autosomal dominant familial ALS (FALS) are clinically similar (Mulder et al. Neurology, 36:511-517, 1986; Swerts et al., Genet. Hum, 24:247-255, 1976; Huisquinet et al., Genet. 18:109-115, 1980). It has been shown that in some but not all FALS pedigrees the disease is linked to a genetic defect on chromosome 21q (Siddique et al., New Engl. J. Med., 324:1381-1384, 1991.
Parkinson""s disease (paralysis agitans) is a common neurodegenerative disorder which appears in mid to late life. Familial and sporadic cases occur, although familial cases account for only 1-2 percent of the observed cases. The neurological changes which cause this disease are somewhat variable and not fully understood. Patients frequently have nerve cell loss with reactive gliosis and Lewy bodies in the substantia nigra and locus coeruleus of the brain stem. Similar changes are observed in the nucleus basalis of Meynert. As a class, the nigrostriatal dopaminergic neurons seem to be most affected.
The disorder generally develops asymmetrically with tremors in one hand or leg and progresses into symmetrical loss of voluntary movement. Eventually, the patient becomes incapacitated by rigidity and tremors. In the advanced stages the disease is frequently accompanied by dementia.
Diagnosis of both familial and sporadic cases of Parkinson""s disease can only be made after the onset of the disease. Anticholinergic compounds, propranolol, primidone and levodopa are frequently administered to modify neural transmissions and thereby suppress the symptoms of the disease, though there is no known therapy which halts or slows the underlying progression. Deprenyl has shown some therapeutic promise.
Huntington""s disease is a progressive disease which is always transmitted as an autosomal dominant trait. Individuals are asymptomatic until the middle adult years, although some patients show symptoms as early as age 15. Once symptoms appear, the disease is characterized by choreoathetotic movements and progressive dementia until death occurs 15-20 years after the onset of the symptoms.
Patients with Huntington""s disease have progressive atrophy of the caudate nucleus and the structures of the basal ganglia. Atrophy of the caudate nucleus and the putamen is seen microscopically where there is an excessive loss of neural tissue. However, there are no morphologically distinctive cytopathological alterations which have been observed.
Although some of the characteristic mental depression and motor symptoms associated with Huntington""s may be suppressed using tricyclic antidepressants and dopamine receptor antagonists, respectively, no therapy exists for slowing or preventing of the underlying disease process. Huntington""s disease appears to map to a single locus on chromosome 4 and a linkage test currently exists for the clinical assessment of disease risk in presymptomatic individuals with afflicted relatives.
Hallervorden-spatz disease is a neurodegenerative disease which affects the neurons in the region of the basal ganglia. The symptoms generally appear during childhood and adolescence and the disease appears with an inheritance pattern that appears to be autosomal recessive. Patients show abnormalities in muscle tone and movement such a choreoathetosis and dystonia similar to that seen in parkinsonism. As the disease progresses there is increasing dementia. Death generally occurs approximately ten years after onset.
There is no known presymptomatic diagnosis, cure or treatment for Hallervorden-Spatz disease. However, iron toxicity has recently been implicated in the progression of this disease Greenfield, Neuropathology, W. Blackwood and J. A. N. Corsellis, Eds. (Edinborgh; T. and A. Constable, Ltd., 1976) pages 178-180. As a result of this implication, the chelating agent deferoxamine mesylate has been administered to patients. However, this therapeutic approach has shown no definite benefit (Harrison""s Principles of Internal Medicine, Wilson et al. Eds., McGraw-Hill, Inc., New York, 1991).
Alzheimer""s disease is the most important of the neurodegenerative diseases due to the high frequency of occurrence within the population and the fatal course of the disease. Two forms of the disease exist: presenile dementia, in which the symptoms emerge during middle age, and senile dementia which occurs in the elderly. Both forms of the disease appear to have the same pathology. A clear genetic predisposition has been found for presenile dementia. Familial autosomal dominant cases have been reported and the majority of individuals with trisomy 21 (Down""s syndrome) develop presenile dementia after the age of 40. The familial Alzheimer""s cases map to chromosomes 14, 19 and 21, with more than one locus on 21.
Olivopontocerebellar atrophy (OPCA) is a disease classification which includes a number of disorders characterized by a combination of cerebellar cortical degeneration, atrophy of the inferior olivary nuclei and degeneration and disappearance of the pontine nuclei in the basis pontis and middle cerebellar peduncles. Autosomal dominant inheritance is characteristic in most families. In one family, termed the Schut family, genetic linkage has been shown to chromosome 6. An excess of glutamate has been implicated as the causative agent in this disease. A gene with an expanded CAG trinucleotide repeat [causes one form of OCPA] ha snow been identified and eluted sequencing can be used for diagnosis (Orr et al., Nature Genetics 4:221-226, 1993).
The human superoxide dismutases are actually at least three different enzymes: cytosolic Cu/Zn superoxide dismutase encoded by the SOD1 gene on chromosome 21 (Levanon et al., EMBO J. 77-84, 1985 and Hjalmarsson et al., P.N.A.S. 84:6340-6344, 1987); mitochondrial superoxide dismutase encoded by the SOD2 gene on chromosome 6 (Wispe et al., Biochim. Biophys. Acta. 994:30-36, 1989); and extracellular superoxide dismutase encoded by the SOD3 gene on chromosome 4 (Hjalmarsson, supra). SOD1, for example, is a homodimeric metalloenzyme that catalyzes the dismutation of the toxic superoxide anion O2xe2x80x94 to O2 and H2O2. The major function of the superoxide dismutase is to eliminate O2- resulting from aerobic respiration. As a class of polypeptides present in most living organisms, these enzymes are differentially associated with different metals including iron, manganese, copper and copper-zinc.
In Guam an inherited disease termed Parkinsonism-dementia complex has been described. Clinical, pathological and familial studies have indicated that this disease is a clinical variant of the local form of ALS. Cases of presenile dementia in the absence of ALS or Parkinsonism have also been observed in this population (Kurland et al. In Norris FH Jr. and Kurland LT eds. Motor Neuron Diseases: Research on amyotrophic lateral sclerosis and related disorders. NY: Grune and Stratton, 1969; 84:28-50; Hirano et al., Brain 84:642-661, 1961; and Hirano et al., Brain 84:662-679, 1961).
Hallewell et al. (U.S. Pat. No. 5,066,591) describe methods and compositions for the production of human copper/zinc superoxide dismutase polypeptides in microorganisms.
Hallewell (U.S. Pat. No. 5,084,390) describe recombinant Cu/Zn superoxide dismutase polymers having an extended in vivo half-life composed of SOD monomers covalently coupled to each other.
Bruice (International Patent Application No. PCT/US91/06558) describe synthetic enzymes that mimic catalytic activity of superoxide dismutase.
Bracco et al. (P.S.E.B.M. 196:36-41, 1991) have measured the levels of superoxide dismutase in the cerebral spinal fluid of patients with age-related neurodegenerative disorders including ALS, Alzheimer""s disease, and a reference group of normal subjects. Bracco et al. report that the superoxide dismutase activity was found to increase with the age of the subject while no significant correlation was found in the ALS and Alzheimer""s disease patients. The activity mean values were found to be significantly lower in patients with ALS and Alzheimer""s disease.
Liu et al. (Amer. Physiol. Soc. H589-H593, 1989) describe the administration of polyethylene glycol-conjugated superoxide dismutase and catalase to reduce ischemic brain injury in rats.
Olanow (Ann Neurol. 32:52-59, 1992) have proposed free radicals as the cause of neuronal injury in several neurological disorders, including Parkinson""s disease and ischemic brain injury.
We have discovered that mutations in superoxide dismutase cause familial amylotrophic lateral sclerosis. Accordingly we have determined methods for the diagnosis and treatment of amyotrophic lateral sclerosis and other cell death disease, particularly neurodegenerative diseases. Methods are provided for treating familial amyotrophic lateral sclerosis and amyotrophic lateral sclerosis as well as other cell death diseases which are the result of decreased SOD activity, altered SOD enzymatic activity, and altered SOD physical characteristics. In addition, therapeutics for diseases caused by alterations in the SOD biochemical pathway are provided.
In the first aspect, the invention features methods of diagnosing an increased likelihood of developing cell death disease in a patient. The methods include analyzing the DNA of the patient to determine whether the DNA contains a mutation in SOD coding sequence, such a mutation being an indication that the patient has an increased likelihood of developing a cell death disease. The methods may be used to diagnose a cell death disease, particularly neurodegenerative disease, more particularly Parkinson""s disease, Huntington""s disease, Alzheimer""s disease, Hallervorden-Spatz disease, olivopontocerebellar atrophy, multiple system atrophy, progressive supranuclear palsy, diffuse lewy body disease, corticodentatonigral degeneration, progressive familial myoclonic epilepsy, strionigral degeneration, torsion dystonia, familial tremor, Gilles de la Tourette syndrome, and Hallervorden-Spatz disease, and ALS which is familial, sporadic typical, or atypical in nature. These methods may also be used for the diagnosis of a SOD related disease in a fetus.
The methods may include amplifying a SOD-encoding gene of the patient using SOD-specific primers, and then analyzing the amplified gene. The DNA may be analyzed by nucleotide sequencing, SSCP analysis, RFMP, heteroduplex analysis or RFLP analysis. The amplifying may be carried out by PCR reaction, by reverse transcriptase PCR or by any other method available to obtain a sufficient amount of DNA.
The primer sequence may be derived from SOD1 nucleic acids, SOD2 nucleic acids, SOD3 nucleic acids or nucleic acids from any other human SOD gene.
Antibodies which recognize familial amyotrophic lateral sclerosis SOD polypeptides but fail to recognize wild-type SOD may also be used for the diagnosis of familial amyotrophic lateral sclerosis in patients.
In the second aspect, the invention features kits for the diagnosis of a cell death disease in a patient. The kits may include one or more SOD gene-specific PCR primers or antibodies recognizing the SOD polypeptides. The PCR primers may include a SOD1-specific nucleic acid sequences, SOD2-specific nucleic acid sequences, SOD3-specific nucleic acid sequences. These kits may be used to diagnose any of the above-referenced diseases.
Kits which include antibodies which specifically recognize mutant SOD polypeptides present in amyotrophic lateral sclerosis patients are part of the kits of the inventor.
In the third aspect, the invention features methods of treating a patient with a disease involving a mutant SOD encoding gene or environmentally induced ALS. The methods include administering to the patient an antioxidant, effective to reduce the symptoms the disease in the patient. The antioxidant may be vitamin C, vitamin E, a lazaroid, BHA, BHT, Beta-carotene, urate, bilirubin, glutathione, dimercaptrol lutein, upiguinol-10, dithiothreotol, mercaptan, a sulfa compound, methionine, cystein, or N-acetyl cysteine, or any other antioxidant which reduces the level of toxic compounds in the affected cells. Most preferably, the antioxidant is vitamin C or vitamin E.
Also included are methods of treating a patient with a disease involving a mutant SOD encoding gene or a patient with sporadic ALS due to environmental causes which include administering to the patient SOD polypeptide, in an amount effective to reduce the symptoms of said disease in said patient. The SOD polypeptide may be SOD Cu/ZnSOD, mSOD, ecSOD, or derivatives, as described below.
Methods of treating the above patients may also include administering to the patient a chelating agent, e.g. desferoxamine, or transgene including a nucleotide sequence encoding a SOD polypeptide e.g., a nucleotide sequence which encodes the Cu/ZnSOD polypeptide, the mSOD polypeptide, or ecSOD polypeptide. Preferably, the nucleotide sequence encodes Cu/ZnSOD or mSOD, and most preferably the nucleotide sequence encodes Cu/ZnSOD.
Also included in the invention is a method for treating a patient with a disease involving a mutant SOD encoding gene. This method includes first identifying a mutant SOD polypeptide-encoding gene in the DNA of the patient, and, second, administering to the patient a therapeutic amount of the anti-sense RNA homolog of a gene encoding a SOD polypeptide. The polypeptide may be wild-type SOD or a polypeptide encoded by the mutant SOD-encoding gene.
Also included is a method for treating a patient with a disease involving a mutant SOD encoding gene, wherein the mutant SOD polypeptide-encoding gene in the DNA is identified in the patient, and a therapeutic amount of a transgene encoding the wild-type homolog of the mutant SOD polypeptide is administered.
Further included is a method for treating a patient with a disease involving a mutant SOD-encoding gene, which comprises identifying the mutant SOD polypeptide-encoding gene in the DNA of the patient, and administering to the patient a therapeutic amount of a transgene encoding the anti-sense homolog of said wild-type SOD RNA.
Also a part of the invention is a method of treating a patient with a disease involving a mutant SOD encoding gene by administering to the patient an antibody which is sufficient to partially inactivate said mutant SOD polypeptide.
A method of treating a patient with a disease involving a mutant SOD encoding gene or a patient with sporadic ALS due to environmental causes either of whose disease is caused at least in part by excess SOD activity by administering to the patient an inhibitor of wild-type SOD, such as those provided herein, is a part of the invention.
A method of treating a patient with a disease involving a deleterious mutant SOD encoding gene or a patient with sporadic ALS due to environmental causes by the administering of a mutant SOD polypeptide with increased SOD enzymatic activity compared to wild-type SOD is also included as a part of the invention. Such a patient may also be treated by administering a nucleotide sequence encoding a non-wild-type therapeutic SOD polypeptide mutant different from and capable of inhibiting the deleterious SOD polypeptide. As in all methods, this SOD polypeptide may be a fragment of SOD, an analog of SOD, or a non-peptide mimetic of SOD.
Further included is a method of treating a patient with a disease involving a mutant SOD encoding gene by administering to the patient a compound which participates in a biochemical pathway involving a SOD polypeptide. These compounds may include glutathione peroxidase, catalase, or nitric oxide synthase. Specifically, peroxide-reducing polypeptides may be administered, as described below.
The invention also includes methods of treating or preventing ALS and FALS by the administration of inhibitors and agonists of SOD. This method is appropriate in patients in whom SOD gene which encodes a polypeptide which confers altered or increased SOD enzymatic activity. The chelating agent may be disferoxamine, EDTA, EGTA, DETC, BCDA, penicillamine, tetracycline, a metallothionein protein or an apo-metal binding protein. The metallothionein protein may be a yeast copper metallotheonein. The apo-metal binding protein may be one or more of apo-superoxide dismutase, hemoglobin, myoglobin, or plastocyanin.
Any of the methods described herein may be used alone or in combination. For example, it may be desirable to administer one or more antioxidants in combination with a chelating agent and or a SOD polypeptide therapeutic.
Any of the following diseases may be treated using one or more of the above methods: a cell death disease, particularly a neurodegenerative disease, more particularly Parkinson""s disease, Huntington""s disease, Alzheimer""s disease, Hallervorden-Spatz disease, olivopontocerebellar atrophy, multiple system atrophy, progressive supranuclear palsy, diffuse lewy body disease, corticodentatonigral degeneration, progressive familial myoclonic epilepsy, strionigral degeneration, torsion dystonia, familial tremor, Gilles de la Tourette syndrome, and ALS which is familial, sporadic typical, or atypical in nature.
In a fourth aspect, the invention features antibodies reactive with a FALS polypeptide but not significantly reactive with a wild-type SOD polypeptide. These antibodies may be monoclonal or polyclonal and may be obtained by subtractive techniques. The antibodies may be sufficient for the inactivation of a SOD polypeptide.
These antibodies may be used as stated above to diagnose ALS in a patient by contacting a blood sample of said patient with the antibody.
In a fifth aspect, the invention features methods of treating a patient with a neoplasm by administering to the patient a FALS polypeptide. A patient with a neoplasm, may also be treated by the administration of transgene encoding an FALS polypeptide.
In the sixth aspect, the invention features a transgenic non-human animal whose somatic and germ cells contain a transgene for a disease-causing mutant SOD polypeptide having a nucleic acid sequence encoding a disease causing SOD polypeptide in an expressible genetic construction. The animal may be a mouse, a worm, or any other animal useful for research or drug development.
In the seventh aspect, the invention features a bacterial or yeast cell containing purified nucleic acid derived from a FALS gene.
The eighth aspect, the invention features purified DNA encoding a purified FALS polypeptide, purified RNA encoding a purified FALS polypeptide, and purified FALS polypeptide.
A ninth aspect of the invention is the use of any of the methods or compounds of the invention which do not solely depend upon the physical properties of a mutant SOD polypeptide for the treatment of a disease of cell death which is the result of a mutation or imbalance in a component of the SOD pathway other than the SOD polypeptide. For example, treatment of diseases due to defects in the production or function of glutathione peroxidase, catalase and nitric oxide synthase. Methods useful for the treatment of these disorders include administration of wild-type and mutant SOD, anti-sense RNA to SOD encoding sequences, use of antibodies to wild-type SOD, and use of analogs and inhibitors of compounds in the SOD pathway.
More specifically, the invention provides therapies using Cu/Zn superoxide dismutase (Cu/ZnSOD), mitochondrial superoxide dismutase (mSOD), or extracellular superoxide dismutase (ecSOD) (FIGS. 2-4 and SEQ ID NOS: 1-3, respectively), as well as other naturally occurring superoxide dismutase polypeptides. Also included are: allelic variations; natural mutants; induced mutants; proteins encoded by DNA that hybridizes under high (e.g., washing at 2xSSC at 40 C with a probe length of at least 40 nucleotides) stringency conditions to naturally occurring Cu/Zn SOD, mSOD, or ecSOD-encoding nucleotide sequences, i.e. SOD1, SOD2, or SOD3; for other definitions of high and low stringency see Current Protocols in Molecular Biology, John Wiley and Sons, New York, 1989, 6.3.1-6.3.6, hereby incorporated by reference). The term xe2x80x9cSOD polypeptidexe2x80x9d also includes chimeric polypeptides that include Cu/ZnSOD, mSOD, or ecSOD together with unrelated sequences.
The invention also includes any biologically active fragment or analog of Cu/ZnSOD, mSOD or ecSOD. By. xe2x80x9cbiologically activexe2x80x9d is meant possessing therapeutically useful superoxide reducing activity which is characteristic of the Cu/ZnSOD, mSOD, or ecSOD polypeptides shown in FIGS. 2-4 (SEQ ID NOS: 1-3). Therapeutically useful activity of a Cu/ZnSOD, mSOD or ecSOD fragment or Cu/ZnSOD, mSOD, or ecSOD analog, can be determined in any of a variety of Cu/ZnSOD, mSOD or ecSOD assays. For example, those assays described in Wayne and Fridovich (Analytical Biochemistry, 161: 559-566 (1987)), McCord and Fridovich (J. of Biol. Chem., 244: 6049-6055 (1969)), and Salin and McCord (J. of Clin. Invest., 54:1005-1009 (1974)) may be used to determine superoxide dismutase activities of Cu/ZnSOD, mSOD or ecSOD. A Cu/ZnSOD, mSOD or ecSOD analog possessing, most preferably 90%, preferably 40%, or at least 10% of the activity of a wild-type or mutant Cu/Zn SOD, mSOD, or ecSOD polypeptide (shown in FIGS. 2-4; SEQ ID NOS: 1-3), in any in vivo or in vitro Cu/ZnSOD, mSOD or ecSOD assay (e.g., those described herein) is considered biologically active and useful in the methods of the invention.
Preferred analogs include 155-amino acid Cu/Zn SOD, 222 amino acid mSOD, or 240 amino acid ecSOD (or biologically active fragments thereof) whose sequences differ from the wild-type sequence only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not destroy the polypeptide""s relevant biological activity as measured using in vivo or in vitro (e.g., those described above). Preferred analogs also include Cu/ZnSOD, mSOD, or ecSOD (or biologically active fragments thereof) which are modified for the purpose of increasing peptide stability; such analogs may contain, for example, one or more desaturated peptide bonds or D-amino acids in the peptide sequence.
Analogs can differ from naturally occurring Cu/ZnSOD, mSOD, or ecSOD polypeptides by amino acid sequence differences or by modifications that do not involve sequence, or by both. Analogs useful for the methods of the invention will generally exhibit at least 65%, more preferably 80%, even more preferably 90%, and most preferably 95% or even 99%, homology with all or part of a naturally occurring Cu/ZnSOD, mSOD, or ecSOD sequence. The length of comparison sequences will generally be at least about 15 amino acid residues, preferably more than 40 amino acid residues. Modifications include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, glycosylation, or carboxylation. Also embraced are versions of the same primary amino acid sequence that have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine. Analogs can differ from naturally occurring Cu/ZnSOD, mSOD, or ecSOD polypeptides by alterations of their primary sequence. These include genetic variants, both natural and induced. Also included are analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., xcex2 or xcex3 amino acids. Alternatively, increased stability may be conferred by cyclizing the peptide molecule.
The invention also provides methods of using SOD polypeptides (or nucleotide sequences encoding polypeptides) which are obtained from other living organisms which are found to synthesize superoxide dismutases, e.g., E.coli, Saccharomyces cerevisiae, and C. elegans. Useful mutants of such SOD polypeptides are those which have increased stability or other desirable properties.
The invention also includes therapeutic uses of polypeptides (or nucleotide sequences encoding polypeptides) which are substantially (at least 70%) homologous to wild-type SOD polypeptides or genes. xe2x80x9cHomologousxe2x80x9d refers to the sequence similarity between two polypeptides or nucleic acids. When a position in both of the two compared sequences is occupied by the same base or amino acid monomeric subunit, e.g., if a position in each of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences. For example, if 6 of 10 of the positions in two sequences are homologous, then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC are 50% omologous.
The invention also includes uses of the compounds described herein in the manufacture of medicaments to be used in the diagnosis and treatment of patients with any of the cell death diseases.
Substantially pure Cu/ZnSOD, mSOD, and ecSOD polypeptides can be produced in quantity using standard recombinant DNA-based techniques. Thus, recombinant Cu/ZnSOD, mSOD2, or ecSOD polypeptides can be administered with a pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage form.
Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the polypeptide to patients suffering from or presymptomatic for a disease of cell death.
Further included as an aspect gene of the invention are the FALS-SOD polypeptides, e.g., those polypeptides encoded by the nucleic acid of patients with FALS due to a SOD mutation. Also included are the nucleic acids which encode these mutant polypeptides. Also included as an aspect of the invention are antibodies, particularly monoclonal antibodies, which are reactive with FALS-SOD polypeptides.
In addition to substantially full-length polypeptides, the invention also includes biologically active fragments of the polypeptides. As used herein, the term xe2x80x9cfragmentxe2x80x9d, as applied to a polypeptide, will ordinarily be at least about 10 contiguous amino acids, typically at least about 20 contiguous amino acids, more typically at least about 30 contiguous amino acids, usually at least about 40 contiguous amino acids, preferably at least about 50 contiguous amino acids, and most preferably at least about 60 to 80 or more contiguous amino acids in length. Fragments of Cu/ZnSOD, MSOD, or ecSOD can be generated by methods known to those skilled in the art. The ability of a candidate fragment to exhibit a biological activity of Cu/ZnSOD, mSOD, or ecSOD can be assessed by methods described below. Also included are Cu/ZnSOD, mSOD, or ecSOD polypeptides containing amino acids that are normally removed during protein processing (for example, the leader sequence of ecSOD), including additional amino acids that are not required for the biological activity of the polypeptide, or including additional amino acids (if any) that result from alternative mRNA splicing or alternative protein processing events.
The invention also provides methods of using SOD polypeptides (or nucleotide sequences encoding polypeptides) which are obtained from other living organisms which are found to synthesize superoxide dismutases, e.g., E.coli, Saccharomyces cerevisiae, and C. elegans. Useful mutants of such SOD polypeptides are those which have increased stability or other desirable properties.
The invention also includes therapeutic uses of polypeptides (or nucleotide sequences encoding polypeptides) which are substantially (at least 70%) homologous to wild-type SOD polypeptides or genes. xe2x80x9cHomologousxe2x80x9d refers to the sequence similarity between two polypeptides or nucleic acids. When a position in both of the two compared sequences is occupied by the same base or amino acid monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The homology between two sequences is a function of the number of matching or homologous positions share by the two sequences. For example, if 6 of 10 of the positions in two sequences are homologous, then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC are 50% homologous.
Substantially pure Cu/ZnSOD, mSOD, and ecSOD polypeptides can be produced in quantity using standard recombinant DNA-based techniques. Thus, recombinant Cu/ZnSOD, mSOD2, or ecSOD polypeptides can be administered with a pharmaceutically acceptable diluent, carrier, or excipient, in unit dosage form.
Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the polypeptide to patients suffering from or presymptomatic for a disease of cell death. A xe2x80x9csubstantially purexe2x80x9d preparation of a polypeptide is a preparation which is substantially free (e.g., to the extent required for formulating Cu/ZnSOD, mSOD2, or ecSOD into a therapeutic composition) of the proteins with which it naturally occurs in a cell.
The formulations of the invention can be administered for example, by parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, ihtranasal, aerosol, or oral administration.
Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
Methods well known in the art for making formulations are to be found in, for example, xe2x80x9cRemington""s Pharmaceutical Sciencesxe2x80x9d. Formulations for parenteral administration may, for example, contain excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of present factors. Other potentially useful parenteral delivery systems for the factors include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
Toxic SOD mutants and nucleotide sequences encoding such polypeptides can be formulated by any of the above methods for use as therapies for diseases of cell proliferation, e.g., cancer.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
The drawings will first be briefly described.